Mycobacterium tuberculosis, the bacterium responsible for tuberculosis disease, is an intracellular pathogen that survives and grows within macrophages. Proteins secreted or localized to the bacterial cell surface by M. tuberculosis can interact with host cells, and they contribute to pathogenesis and the immune response. Many of the proteins in this subcellular category remain to be identified or studied. Further, we hypothesize that there is an important class of secreted and surface proteins - those exclusively exported while M. tuberculosis is intracellular - which are missed by current detection methods. We recently established a genetic reporter system in which beta-lactamase enzymes are used in protein fusions to report on protein export directly in a beta-lactam sensitive mutant of M. tuberculosis. Because beta-lactam antibiotics target cell wall synthesis enzymes, beta-lactamases must be exported out of the cytoplasm to protect the bacterium. We believe our beta-lactamase reporter system has a novel capability of identifying proteins secreted during intracellular growth of M. tuberculosis in beta-lactam treated macrophages. In this RO3 application we propose to use this reporter in a genetic selection scheme to identify proteins exported by M. tuberculosis during growth in the host environment. A subset of the proteins identified will be further studied for roles in intracellular growth in macrophages. This research will complement existing genomic approaches and uncover new virulence factors and candidate antigens that should aid development of novel disease intervention and diagnostic strategies. [unreadable] [unreadable] [unreadable]